Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
Gamma glutamyl hydrolase (GH) (EC 3.4.22.12) catalyzes the hydrolysis of the polyglutamate side-chain folyl polyglutamates and antifolyl polyglutamates (Stokstad and Koch 1967; Elsenhans et al. 1984; Chandler et al. 1986; Silink et al. 1975; Horne et al. 1981; Samuels et al. 1986; Bhandari et al. 1990; Rosenberg and Neuman 1974; Priest et al. 1982; Saini and Rosenberg 1974; Shin et al. 1974; McGuire and Coward 1984; Wang et al. 1986; Rao and Noronha 1977; Reisenauer et al. 1977; Wang et al. 1993). Gamma glutamyl hydrolase has been characterized from a number of sources, and it exhibits either endo- or exo-peptidase activity, depending upon the tissue of origin (Stokstad and Koch 1967; Elsenhans et al. 1984; Silink et al. 1975; Horne et al. 1981; Samuels et al. 1986; Bhandari et al. 1990; Rosenberg and Neuman 1974; Priest et al. 1982; Saini and Rosenberg 1974; Shin et al. 1974). In many tissues the enzyme is lysosomal with an acidic pH optimum (McGuire and Coward 1984). In addition, the enzyme is often sulfhydryl- and Zn.sup.2+ -dependent (Chandler et al. 1986; Silink et al. 1975; Horne et al. 1981; Bhandari et al. 1990; McGuire and Coward 1984; Wang et al. 1986; Rao and Noronha 1977; Wang et al. 1993), appears to be a glycoprotein in many cases (Silink et al. 1975; Wang et al. 1993), and has a reported molecular weight of 50 to 150 kDa (Elsenhans et al. 1984; Chandler et al. 1986; Silink et al. 1975; Priest et al. 1982; McGuire and Coward 1984; Rao and Noronha 1977; Reisenauer et al. 1977; Wang et al. 1993). A thorough analysis of the mechanism of this unique peptidase that cleaves only gamma glutamyl linkages is not available and a knowledge of the detailed structure of the protein and the gene encoding it are not yet delineated.
It has been shown that resistance to the antifolate 5,10-didiazatetrahydrofolate can be acquired by enhancement of GH enzyme activity in rat H35 hepatoma cells (Rhee et al. 1993). In addition, GH is hormonally controlled, with both insulin and estrogen altering its activity in responsive cell lines and tissues (Wang et al. 1993; Galivan and Rhee 1995; Krumdieck et al. 1975). For many years it was thought that GH was a lysosomal enzyme, but recent studies using cell culture systems have shown that while its intracellular location is primarily the lysosome, most of the enzyme activity is secreted, a feature that appears thus far to be universal in neoplastic cells (O'Connor et al. 1991; Yao et al. 1995; Rhee et al. 1995).
The availability of the cDNA and amino acid sequence for GH and a polyclonal antibody to the protein offers the possibility of investigating a number of questions concerning this enzyme. The role of GH in the cellular metabolism of folylpolyglutamate coenzymes and in the cytotoxic activity of antifolates can be evaluated in detail. GH activity is known to be altered by a number of factors including insulin (Galivan and Rhee 1995), estrogen (Krumdieck et al. 1975), and selection for resistance with 5,10-didiazatetrahydrofolate in rat (Rhee et al. 1993; Yao et al. 1995) and human (Pizzorno et al. 1995) cell lines. With the availability of molecular and immunological probes, the mechanism of alterations in GH activity can be investigated. The cellular trafficking of the glycoprotein can be approached with an emphasis on the mechanism and significance of secretion. These probes could be used to evaluate the abundant secretion of GH by human breast cancer cell lines in culture (Rhee et al. 1995), which is potentially related to the high levels of GH in serum of metastatic breast cancer patients (Baggott et al. 1987). In addition, relatively large amounts of enzyme could become available for the first time when an appropriate expression system is established, and this will allow detailed analysis of the structure and mechanism of GH.
A need continues to exist, therefore, for the determination of the nucleotide and amino acid sequences of gamma glutamyl hydrolase.